530
Letters to the Editor
Low-dose dipyridamole treatment partially prevents diabetes mellitus-induced vascular endothelial and renal abnormalities in rats Arun Kumar Sharma a, Deepa Khanna a,⁎, Pitchai Balakumar b a b
Department of Pharmacology, Rajendra Institute of Technology and Sciences (RITS), Sirsa 125 055, India Pharmacology Unit, Faculty of Pharmacy, AIMST University, 08100 Bedong, Malaysia
a r t i c l e
i n f o
Article history: Received 9 January 2014 Accepted 12 January 2014 Available online 23 January 2014 Keywords: Diabetes mellitus Low-dose dipyridamole PI3-K eNOS Vascular endothelial dysfunction Nephropathy
Dipyridamole is a conventionally used platelet inhibitor employed for the secondary prevention of transient ischemic attack. Dipyridamole could potentiate some of the vascular protective effects of nitric oxide (NO) [1]. Intriguingly, the vascular protective action of dipyridamole was reported to be mediated by its stimulatory action on endothelial nitric oxide synthase (eNOS) activity [2]. However, the effect of dipyridamole in diabetes mellitus-induced vascular endothelial dysfunction (VED) is not yet known. There are strong associations between diabetic VED and nephropathy [3,4]. Administration of dipyridamole either alone or in combination with aspirin reduced the incidence of proteinuria in patients with diabetic nephropathy [5]. Dipyridamole at high-dose is considered proischemic, and it could cause a marked coronary steal effect. Dipyridamole, however, at lowdose orally might have a minimal hemodynamic effect [6]. The present study investigated the effect of low-dose dipyridamole in diabetes mellitus-induced VED and nephropathy in rats. The experimental protocol of this study was approved by the ‘Institutional Animal Ethics Committee’. Wistar albino rats of either sex (n = 6) weighing about 250 to 350 g were used. Group I (Normal Control), rats were maintained on standard food and water, and no treatment was given. Group II (Diabetic Control), rats were administered streptozotocin (STZ) (50 mg/kg, i.p., once), and were allowed for 8 weeks to develop experimental VED and nephropathy (rats showing blood glucose level of greater than 200 mg/dl after 72 h of STZ administration were selected and named as diabetics). Group III
(Dipyridamole per se), normal rats were administered low-dose dipyridamole (30 mg/kg/day, p.o.) for 4 weeks. Group IV (Dipyrida), the diabetic rats after 4 weeks of STZ-administration were treated with low-dose dipyridamole (30 mg/kg/day, p.o.) for 4 weeks. Groups V and VI (Wortmannin or L-NAME Incubated Aortic Ring of Dipyridamole Treated), the diabetic rats were treated with low-dose dipyridamole (30 mg/kg/day, p.o., 4 weeks) as mentioned in group IV. The in vitro effects of wortmannin (100 nM) or L-NAME (100 μM) (the aortic ring was incubated with either wortmannin or L-NAME for 30-min) on endothelium-dependent and -independent relaxation in the phenylephrine (3 × 10− 6 M)-precontracted isolated aortic ring preparation were evaluated. One way ANOVA, followed by Tukey's multiple comparison test was employed to analyse the data. STZ-induced diabetic rats developed VED and nephropathy in 8 weeks. Diabetes mellitus-induced VED was associated with marked reduction in acetylcholine-induced endothelium-dependent relaxation, decrease in aortic and serum nitrite/nitrate concentration, and induction of oxidative stress and lipid alteration. Scanning electron microscopic and histological studies on thoracic aorta revealed a marked impairment in vascular endothelial integrity. Moreover, elevation of serum creatinine and blood urea nitrogen, and induction of proteinuria accompanying with renal oxidative stress were noted in diabetic rats. However, treatment with low-dose dipyridamole partially, but substantially prevented aforementioned vascular endothelial and renal abnormalities in diabetic rats without affecting the elevated glucose concentration. Interestingly, incubation of aortic rings, isolated from dipyridamole-treated diabetic rats, with either wortmannin (inhibitor of phosphatidylinositide 3-kinase, PI3-K) or LNAME (inhibitor of nitric oxide synthase, NOS) markedly abolished dipyridamole-associated improvement in acetylcholine-induced endothelium-dependent relaxation (Table 1, Figs. 1 and 2). To summarize, dipyridamole in low-dose partially, but significantly prevents diabetes mellitus-induced VED by activating endothelial PI3K-eNOS-NO signalling pathways. The renovascular reduction of oxidative stress might explain the mechanism pertaining to the partial renoprotective action of low-dose dipyridamole in diabetes mellitus. We express our gratitude to the management of RITS, Sirsa, Haryana, India for the support. The authors of this manuscript have certified that
Table 1 Effect of low-dose dipyridamole on serum glucose, total cholesterol (TC) and kidney weight/body weight ratio (KW/BW) in diabetic rats. Assessments
Normal Control
Diabetic Control
Dipyridamole per se
Dipyridamole Treated
Serum glucose (mg/dl) Serum TC (mg/dl) KW/BW (mg/g)
92.26 ± 6.39 53.55 ± 4.52 4.8 ± 0.44
277.47 ± 30.23a 95.87 ± 6.76a 6.88 ± 0.23a
96.45 ± 8.27 55.13 ± 5.32 4.7 ± 0.16
272.94 ± 18.67 65.78 ± 9.42b 5.38 ± 0.63b
⁎ Corresponding author at: Department of Pharmacology, RITS, Sirsa 125 055, India. E-mail address: [email protected] (D. Khanna).
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0
Normal Control
Diabetic Control
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Normal Control
Wortmannin Incubated Aortic Ring of Dipyridamole Treated
L-NAME Incubated Aortic Ring of Dipyridamole Treated
Dipyridamole per se
Diabetic Control
Dipyridamole Treated
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40
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Diabetic Control
Dip per se
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Wortmannin Incubated Aortic Ring of Dipyridamole Treated
L-NAME Incubated Aortic Ring of Dipyridamole Treated
C
D
e
Fig. 1. Effect of low-dose dipyridamole on acetylcholine (Ach)-induced endothelium-dependent (a) and sodium nitroprusside (SNP)-induced endothelium independent (b) relaxation of aorta (in all figures and in the table, all values were represented as mean ± S.D., a = p b 0.05 vs Normal Control; b = p b 0.05 vs Diabetic Control; c = p b 0.05 vs Dipyridamole Treated), on aortic nitrite/nitrate concentration (c), on serum nitrite/nitrate (d), on the integrity of vascular endothelium of aorta (e), the scanning electron microscopic study was performed to examine the integrity of vascular endothelium of the rat thoracic aorta using Zeiss EVOMA-10 scanning electron microscope (scale bar = 20 μm). (A) Normal Control; (B) Diabetic Control; (C) Dipyridamole per se; (D) Dipyridamole Treated, on the integrity of vascular endothelial layer of aorta (f). The histological examination was performed using Motic Microscope BA310 (Motic, USA) in diabetic rats.
they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. References [1] Kim HH, Liao JK. Translational therapeutics of dipyridamole. Arterioscler Thromb Vasc Biol 2008;28:39–42. [2] Venkatesh PK, Pattillo CB, Branch B, et al. Dipyridamole enhances ischaemiainduced arteriogenesis through an endocrine nitrite/nitric oxide-dependent pathway. Cardiovasc Res 2010;85:661–70.
[3] Balakumar P, Chakkarwar VA, Singh M. Ameliorative effect of combination of benfotiamine and fenofibrate in diabetes-induced vascular endothelial dysfunction and nephropathy in the rat. Mol Cell Biochem 2009;320:149–62. [4] Balakumar P, Chakkarwar VA, Krishan P, Singh M. Vascular endothelial dysfunction: a tug of war in diabetic nephropathy? Biomed Pharmacother 2009;63:171–9. [5] Khajehdehi P, Roozbeh J, Mostafavi H. A comparative randomized and placebocontrolled short-term trial of aspirin and dipyridamole for overt type-2 diabetic nephropathy. Scand J Urol Nephrol 2002;36:145–8. [6] Ye Y, Lin Y, Perez-Polo R, et al. Enhanced cardioprotection against ischemiareperfusion injury with a dipyridamole and low-dose atorvastatin combination. Am J Physiol Heart Circ Physiol 2007;293:813–8.
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Normal Control
Diabetic Control
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Diabetic Control
Dipyridamole per se
Dipyridamole per se
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Dipyridamole Treated
Dipyridamole per se Dipyridamole Treated
Fig. 2. Effect of low-dose dipyridamole on serum TBARS (a), on serum creatinine (b), on blood urea nitrogen (c), on microproteinuria (d) and on renal TBARS (e) in diabetic rats.
0167-5273/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcard.2014.01.053
Letters to the Editor
Low-dose dipyridamole treatment partially prevents diabetes mellitus-induced vascular endothelial and renal abnormalities in rats Arun Kumar Sharma a, Deepa Khanna a,⁎, Pitchai Balakumar b a b
Department of Pharmacology, Rajendra Institute of Technology and Sciences (RITS), Sirsa 125 055, India Pharmacology Unit, Faculty of Pharmacy, AIMST University, 08100 Bedong, Malaysia
a r t i c l e
i n f o
Article history: Received 9 January 2014 Accepted 12 January 2014 Available online 23 January 2014 Keywords: Diabetes mellitus Low-dose dipyridamole PI3-K eNOS Vascular endothelial dysfunction Nephropathy
Dipyridamole is a conventionally used platelet inhibitor employed for the secondary prevention of transient ischemic attack. Dipyridamole could potentiate some of the vascular protective effects of nitric oxide (NO) [1]. Intriguingly, the vascular protective action of dipyridamole was reported to be mediated by its stimulatory action on endothelial nitric oxide synthase (eNOS) activity [2]. However, the effect of dipyridamole in diabetes mellitus-induced vascular endothelial dysfunction (VED) is not yet known. There are strong associations between diabetic VED and nephropathy [3,4]. Administration of dipyridamole either alone or in combination with aspirin reduced the incidence of proteinuria in patients with diabetic nephropathy [5]. Dipyridamole at high-dose is considered proischemic, and it could cause a marked coronary steal effect. Dipyridamole, however, at lowdose orally might have a minimal hemodynamic effect [6]. The present study investigated the effect of low-dose dipyridamole in diabetes mellitus-induced VED and nephropathy in rats. The experimental protocol of this study was approved by the ‘Institutional Animal Ethics Committee’. Wistar albino rats of either sex (n = 6) weighing about 250 to 350 g were used. Group I (Normal Control), rats were maintained on standard food and water, and no treatment was given. Group II (Diabetic Control), rats were administered streptozotocin (STZ) (50 mg/kg, i.p., once), and were allowed for 8 weeks to develop experimental VED and nephropathy (rats showing blood glucose level of greater than 200 mg/dl after 72 h of STZ administration were selected and named as diabetics). Group III
(Dipyridamole per se), normal rats were administered low-dose dipyridamole (30 mg/kg/day, p.o.) for 4 weeks. Group IV (Dipyrida), the diabetic rats after 4 weeks of STZ-administration were treated with low-dose dipyridamole (30 mg/kg/day, p.o.) for 4 weeks. Groups V and VI (Wortmannin or L-NAME Incubated Aortic Ring of Dipyridamole Treated), the diabetic rats were treated with low-dose dipyridamole (30 mg/kg/day, p.o., 4 weeks) as mentioned in group IV. The in vitro effects of wortmannin (100 nM) or L-NAME (100 μM) (the aortic ring was incubated with either wortmannin or L-NAME for 30-min) on endothelium-dependent and -independent relaxation in the phenylephrine (3 × 10− 6 M)-precontracted isolated aortic ring preparation were evaluated. One way ANOVA, followed by Tukey's multiple comparison test was employed to analyse the data. STZ-induced diabetic rats developed VED and nephropathy in 8 weeks. Diabetes mellitus-induced VED was associated with marked reduction in acetylcholine-induced endothelium-dependent relaxation, decrease in aortic and serum nitrite/nitrate concentration, and induction of oxidative stress and lipid alteration. Scanning electron microscopic and histological studies on thoracic aorta revealed a marked impairment in vascular endothelial integrity. Moreover, elevation of serum creatinine and blood urea nitrogen, and induction of proteinuria accompanying with renal oxidative stress were noted in diabetic rats. However, treatment with low-dose dipyridamole partially, but substantially prevented aforementioned vascular endothelial and renal abnormalities in diabetic rats without affecting the elevated glucose concentration. Interestingly, incubation of aortic rings, isolated from dipyridamole-treated diabetic rats, with either wortmannin (inhibitor of phosphatidylinositide 3-kinase, PI3-K) or LNAME (inhibitor of nitric oxide synthase, NOS) markedly abolished dipyridamole-associated improvement in acetylcholine-induced endothelium-dependent relaxation (Table 1, Figs. 1 and 2). To summarize, dipyridamole in low-dose partially, but significantly prevents diabetes mellitus-induced VED by activating endothelial PI3K-eNOS-NO signalling pathways. The renovascular reduction of oxidative stress might explain the mechanism pertaining to the partial renoprotective action of low-dose dipyridamole in diabetes mellitus. We express our gratitude to the management of RITS, Sirsa, Haryana, India for the support. The authors of this manuscript have certified that
Table 1 Effect of low-dose dipyridamole on serum glucose, total cholesterol (TC) and kidney weight/body weight ratio (KW/BW) in diabetic rats. Assessments
Normal Control
Diabetic Control
Dipyridamole per se
Dipyridamole Treated
Serum glucose (mg/dl) Serum TC (mg/dl) KW/BW (mg/g)
92.26 ± 6.39 53.55 ± 4.52 4.8 ± 0.44
277.47 ± 30.23a 95.87 ± 6.76a 6.88 ± 0.23a
96.45 ± 8.27 55.13 ± 5.32 4.7 ± 0.16
272.94 ± 18.67 65.78 ± 9.42b 5.38 ± 0.63b
⁎ Corresponding author at: Department of Pharmacology, RITS, Sirsa 125 055, India. E-mail address: [email protected] (D. Khanna).
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Normal Control
Diabetic Control
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Dip per se
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Wortmannin Incubated Aortic Ring of Dipyridamole Treated
L-NAME Incubated Aortic Ring of Dipyridamole Treated
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e
Fig. 1. Effect of low-dose dipyridamole on acetylcholine (Ach)-induced endothelium-dependent (a) and sodium nitroprusside (SNP)-induced endothelium independent (b) relaxation of aorta (in all figures and in the table, all values were represented as mean ± S.D., a = p b 0.05 vs Normal Control; b = p b 0.05 vs Diabetic Control; c = p b 0.05 vs Dipyridamole Treated), on aortic nitrite/nitrate concentration (c), on serum nitrite/nitrate (d), on the integrity of vascular endothelium of aorta (e), the scanning electron microscopic study was performed to examine the integrity of vascular endothelium of the rat thoracic aorta using Zeiss EVOMA-10 scanning electron microscope (scale bar = 20 μm). (A) Normal Control; (B) Diabetic Control; (C) Dipyridamole per se; (D) Dipyridamole Treated, on the integrity of vascular endothelial layer of aorta (f). The histological examination was performed using Motic Microscope BA310 (Motic, USA) in diabetic rats.
they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. References [1] Kim HH, Liao JK. Translational therapeutics of dipyridamole. Arterioscler Thromb Vasc Biol 2008;28:39–42. [2] Venkatesh PK, Pattillo CB, Branch B, et al. Dipyridamole enhances ischaemiainduced arteriogenesis through an endocrine nitrite/nitric oxide-dependent pathway. Cardiovasc Res 2010;85:661–70.
[3] Balakumar P, Chakkarwar VA, Singh M. Ameliorative effect of combination of benfotiamine and fenofibrate in diabetes-induced vascular endothelial dysfunction and nephropathy in the rat. Mol Cell Biochem 2009;320:149–62. [4] Balakumar P, Chakkarwar VA, Krishan P, Singh M. Vascular endothelial dysfunction: a tug of war in diabetic nephropathy? Biomed Pharmacother 2009;63:171–9. [5] Khajehdehi P, Roozbeh J, Mostafavi H. A comparative randomized and placebocontrolled short-term trial of aspirin and dipyridamole for overt type-2 diabetic nephropathy. Scand J Urol Nephrol 2002;36:145–8. [6] Ye Y, Lin Y, Perez-Polo R, et al. Enhanced cardioprotection against ischemiareperfusion injury with a dipyridamole and low-dose atorvastatin combination. Am J Physiol Heart Circ Physiol 2007;293:813–8.
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Dipyridamole per se Dipyridamole Treated
Fig. 2. Effect of low-dose dipyridamole on serum TBARS (a), on serum creatinine (b), on blood urea nitrogen (c), on microproteinuria (d) and on renal TBARS (e) in diabetic rats.
0167-5273/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcard.2014.01.053
